In vitro antiviral activity of polyoxotungstate (PM-19) and other polyoxometalates against herpes simplex virus.

Polyoxotungstates with Keggin-type structure were found to demonstrate marked antiherpetic activity. K7[TiW10PO40].6H2O (PM-19) caused a decrease in plaque formation by several strains of herpes simplex virus (HSV) type 1, including acyclovir-resistant (thymidine kinase-negative) strains, at concentrations which were not toxic to the host cells. The 50% plaque-inhibiting concentration (EC50) for the different strains was between 20 and 50 micrograms/ml. Single-cycle HSV growth was also inhibited by PM-19. PM-19 inhibited viral DNA synthesis in HSV-infected cells at a concentration of 5 micrograms/ml but did not exhibit a virucidal effect, and pretreatment of the host cells with PM-19 did not provide resistance to herpes infection. Yet, virus adsorption to the cells was markedly affected at PM-19 concentrations higher than 25 micrograms/ml. PM-19 was also effective against human cytomegalovirus, but not against adenoviruses and varicella-zoster virus, although it did delay the development of the cytopathic effect of these viruses.     

Virucidal activity of hypericin against enveloped and non-enveloped DNA and RNA viruses.

Hypericin is a polycyclic anthrone first isolated from the plant St. Johnswort and was shown to have dramatic anti-retroviral activity against Friend leukemia virus and radiation leukemia virus in mice. Hypericin displayed marginal activity (IC50 = 6 micrograms/ml) against Moloney murine leukemia virus (Mo-MuLV) in vitro. Hypericin did not display selective antiviral activity against herpes simplex virus, influenza A, adenovirus, or poliovirus. The 50% cytotoxic concentration was approximately 25 micrograms/ml. When virus was incubated with hypericin before infecting cells, the drug was virucidal to all enveloped viruses tested (herpes simplex, influenza virus A, and Mo-MuLV) at concentrations of 1.56 micrograms/ml to 25 micrograms/ml. Hypericin was not virucidal to the non-enveloped viruses tested (adenovirus and poliovirus). These data indicate that the mechanism of viral inactivation for hypericin is dependent upon the presence of a viral lipid envelope. In vivo, hypericin (50 mg/ml) was effective against FLV or HSV-1 if incubated with the virus for 1 h at 37 degrees C before infecting mice, but was not effective if pre-incubated with virus for 1 h at 4 degrees C or if administered concurrently with virus.     

Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture

The antiviral effect of Australian tea tree oil (TTO) and eucalyptus oil (EUO) against herpes simplex virus was examined. Cytotoxicity of TTO and EUO was evaluated in a standard neutral red dye uptake assay. Toxicity of TTO and EUO was moderate for RC-37 cells and approached 50% (TC50) at concentrations of 0.006% and 0.03%, respectively. Antiviral activity of TTO and EUO against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of TTO for herpes simplex virus plaque formation was 0.0009% and 0.0008% and the IC50 of EUO was determined at 0.009% and 0.008% for HSV-1 and HSV-2, respectively. Australian tea tree oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of TTO plaque formation was reduced by 98.2% and 93.0% for HSV-1 and HSV-2, respectively. Noncytotoxic concentrations of EUO reduced virus titers by 57.9% for HSV-1 and 75.4% for HSV-2. Virus titers were reduced significantly with TTO, whereas EUO exhibited distinct but less antiviral activity. In order to determine the mode of antiviral action of both essential oils, either cells were pretreated before viral infection or viruses were incubated with TTO or EUO before infection, during adsorption or after penetration into the host cells. Plaque formation was clearly reduced, when herpes simplex virus was pretreated with the essential oils prior to adsorption. These results indicate that TTO and EUO affect the virus before or during adsorption, but not after penetration into the host cell. Thus TTO and EUO are capable to exert a direct antiviral effect on HSV. Although the active antiherpes components of Australian tea tree and eucalyptus oil are not yet known, their possible application as antiviral agents in recurrent herpes infection is promising.     

Direct inactivation of herpes simplex virus type-2 by rat epidermal protein.

Proteins were extracted from corneocytes of skin of 2-day-old rats and fractionated by gel filtration and cation exchange column chromatography. The different protein fractions were tested for direct inactivation of herpes simplex virus infectivity as determined by reduction of plaque formation. The most effective protein fractions against herpes simplex virus were a neutral pH buffer soluble and mol. wts. ranging from 20 K to 30 K. Amino acid composition of the proteins were virtually identical to epidermal histidine-rich proteins. The activity was significantly (P less than 0.001) stronger against type-2 than type-1. The activity was most stable at pH 7.2 and the rate of inhibition increased in a time-dependent manner up to 4 h. The 50% effective dose was estimated as 1.1 micrograms protein/ml.     

Inhibition of herpes simplex virus type 2 replication in vitro by 1-N-pentadecanoyl-3'-N-trifluoroacetyl kanamycin A

The in vitro antiviral activity as well as the mechanism of action of a new antiviral agent, a kanamycin analogue, 1-N-pentadecanoyl-3'-N-trifluoroacetyl kanamycin A (PTKA) against herpes simplex virus type 2 (HSV-2) was investigated. The drug showed excellent antiviral action with negligible cytotoxic effect on the culture cells. Based on plaque reduction assays the 50% inhibitory dose (ID50) of the drug was 1 microgram/ml, and at 20 micrograms/ml plaque formation was totally suppressed. The compound inhibited viral protein synthesis in infected cells without affecting RNA and DNA synthesis, when added to the cultures after virus adsorption. Moreover, pretreatment of the cells with PTKA before HSV-2 infection, increased the antiviral activity significantly. Dot-blot hybridization analysis revealed that the drug reduced the level of immediate early viral mRNA if applied before infection. There was no detectable action at the level of virus adsorption, penetration or uncoating. These results indicate that PTKA exerted its antiviral action at the early stage of viral replication as well as at the level of viral protein synthesis.     

Thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model

A series of 111 thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds were evaluated as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model. All derivatives tested were potent inhibitors of virus replication with mean 50% inhibitory concentrations of 1.1 micrograms/ml for both type 1 and 2 herpes simplex virus. Inhibitory concentrations for cellular protein and DNA synthesis were considerably higher for many compounds resulting in in vitro therapeutic indices ranging from greater than 100 (highly selective) to less than 1 (negatively selective). All compounds were tested for dermal toxicity following topical administration of saturated solutions in 1,3-butanediol to the shaved, depilated skin of guinea pigs. Approximately 50% of the compounds produced slight to no dermal toxicity whereas the remaining compounds produced moderate to severe dermal toxicity. 28 compounds were evaluated in the cutaneous herpes guinea pig model against herpes simplex virus type 1. A number of N4-monosubstituted 2-acetylpyridine thiosemicarbazones produced highly significant reductions in days to healing and lesion score without producing untoward dermal toxicity. Structure-activity relationships revealed that a reduction of the azomethine bond in the molecule (i.e., conversion of a thiosemicarbazone to a thiosemicarbazide) greatly diminished dermal toxicity apparently without producing a proportional decrease in antiviral activity.     

Anti-herpesvirus activity of citrusinine-I, a new acridone alkaloid, and related compounds

Citrusinine-I, a new acridone alkaloid isolated from the root bark of the citrus plant (Rutaceae), exhibited potent activity against herpes simplex virus (HSV) type 1 and type 2 at low concentrations relative to their cytotoxicity; 50% effective concentrations (ED50) of citrusinine-I were 0.56 micrograms/ml and 0.74 micrograms/ml against HSV-1 and HSV-2, respectively. Inhibitory action was also demonstrated against cytomegalovirus (CMV) and thymidine kinase-deficient or DNA polymerase mutants of HSV-2. The compound markedly suppressed HSV-2 and CMV DNA synthesis at concentrations which did not inhibit the synthesis of virus-induced early polypeptides. However, citrusinine-I had no inhibitory activity against HSV and CMV DNA polymerases in cell-free extracts. Although the target of this inhibitor remains to be elucidated, the most plausible candidate is a virus-coded ribonucleotide reductase. Citrusinine-1, when combined with acyclovir or ganciclovir, synergistically potentiated the antiherpetic activity of these agents. Based on a comparative study of the antiherpetic activity of citrusinine-1 and 28 related compounds, a structure-activity relationship could be established.     

Phenoxazine derivatives inactivate human cytomegalovirus, herpes simplex virus-1, and herpes simplex virus-2 in vitro.

We examined whether phenoxazine derivatives, 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-amino-phenoxazine-3-one (Phx-3) may have antiviral activity against herpes family viruses: human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2). The antiviral activity was evaluated by the selectivity index (SI), which is the ratio of 50% cytotoxic concentration (CC(50)) and 50% antiviral concentration (IC(50)). Among these phenoxazines, Phx-2 exerted strong antiviral activity to HCMV with the SI of 200, while Phx-1 and Phx-3 exerted no marked anti-HCMV activity. Phx-2 also showed moderate inhibition of HSV-1 and HSV-2, with the SI of 6.7 and 17, respectively. In the time-of-addition experiments, inhibitory effect of Phx-2 against HCMV was active even when applied to cells at 100 h after HCMV infection, while ganciclovir (GCV) showed potent inhibition when applied to cells before 42-h post-infection, but its inhibitory effects disappeared thereafter. Attachment and penetration of HCMV was not affected by the presence of Phx-2. When HCMV was pretreated with Phx-2, concentration-dependent virucidal action was observed, suggesting that Phx-2 inactivates HCMV directly. From these data, it was found that Phx-2 might have a different anti-HCMV target from GCV.     

Effect of chlordane on influenza type A virus and herpes simplex type 1 virus replication in vitro

The effect of chlordane on the susceptibility of Madin-Darby canine kidney cells and African Green monkey kidney cells to infection with influenza type A/PR/8/34 (HON1) virus and herpes simplex type 1 virus was determined. Exposure of both cell lines to various concentrations of chlordane for 24 h at 37 degrees C (acute exposure) effected a marked reduction in the efficiency of influenza type A virus infection, except at a dose of 0.025 ppm. Acute exposure of the monkey cells did not alter their susceptibility to herpes simplex virus infection. Viral adsorption studies at 4 and 37 degrees C revealed a marked reduction in the attachment of influenza type A virus to both cell lines following acute exposure to 10 ppm chlordane. Viral inactivation studies carried out at 4 and 37 degrees C failed to reveal differences in the level of influenza type A virus inactivation in the presence or absence of chlordane. Madin-Darby canine kidney cells exposed to 10 ppm chlordane for 60 d (chronic exposure) manifested a decrease in the efficiency of influenza type A virus infection, whereas cells chronically exposed to 0.025 ppm chlordane manifested an increase in the efficiency of influenza type A virus infection relative to mock-treated control cells. When chronically exposed cells were passaged six times in the absence of chlordane, these effects were reversed. Viral adsorption studies carried out at 4 and 37 degrees C on cells chronically exposed to 10 ppm chlordane revealed a decrease in the adsorption of influenza type A virus. Quantitation of the levels of cell-surface sialic acid, the essential terminal sugar on the receptor for influenza type A virus, indicated that the reduced adsorption of influenza type A virus to Madin-Darby canine kidney cells was not due to a loss of cell-surface sialic acid. Our findings indicate that chlordane alters the susceptibility of cells to infection with influenza type A virus but not to herpes simplex type 1 virus.     

Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton

A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5 microg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50 > 1000 microg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 microg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2-400 microg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 degrees C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000-50,000.     

Differential antiviral activity of benzastatin C and its dechlorinated derivative from Streptomyces nitrosporeus

Benzastatin C, a 3-chloro-tetrahydroquinolone alkaloid from Streptomyces nitrosporeus, showed antiviral activity in a dose-dependant manner with EC50 values of 1.92, 0.53, and 1.99 microg/ml against herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and vesicular stomatitis virus (VSV), respectively. In contrast, benzastatin D, the corresponding dechlorinated derivative, did not exhibit any antiviral activity. These results indicate that the antiviral activity of benzastatin C is mediated, in part, due to the chlorine moiety in its molecular structure.     

Antiviral activity of an extract of Cordia salicifolia on herpes simplex virus type 1

A partially purified extract (COL 1-6) from whole plant of Cordia salicifolia showed an inhibitory effect on herpes simplex virus type 1 (HSV-1). The activity of COL 1-6 on different steps of HSV-1 replication in HeLa cells was investigated. Under single-cycle replication conditions, COL 1-6 exerted a greater than 99.9% inhibition in virus yield when added to the cells 3 h or 1.5 h before infection, and even when added 8 h after infection the extract still caused a greater than 99% inhibition. The extract has been shown to have a direct virucidal activity. And also, analysis of early events following infection showed that COL 1-6 affected viral penetration in HeLa cells but did not interfere with adsorption to the cells.     

Inhibition of herpes simplex virus types 1 and 2 replication in vitro by mercurithio analogs of deoxyuridine.

The in vitro antiviral activity of several 5-mercurithio analogs of 2'-deoxyuridine (dUrd) on the replication of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) were examined. Of those compounds tested, the thioglycerol analog of 5-mercuri-2'-deoxyuridine (HgdUrd) was most effective in inhibiting the replication of HSV-1 in KB cells with a 50% inhibitory dose (ID50) of 0.001 micrograms/ml while the glutathione analog of HgdUrd was the most effective in inhibiting the replication of HSV-2 with a ID50 of 0.075 micrograms/ml. Conversely in HeLa TK- cells, the mercaptoguanosine analog of HgdUrd was the most effective compound in inhibiting virus replication with ID50S of 0.098 and 0.001 micrograms/ml for HSV-1 and HSV-2 respectively. These results suggest that these mercurithio analogs of dUrd are as effective as acyclovir in preventing the replication of these herpesviruses.     

Antiviral activity and its mechanism of guanine 7-N-oxide on DNA and RNA viruses derived from salmonid

Guanine 7-N-oxide produced by Streptomyces sp. was found to inhibit in vitro the replication of herpes virus (Oncorhynchus masou virus, OMV), rhabdo virus (infectious hematopoietic necrosis virus, IHNV) and a bi-segmented double-strand virus (infectious pancreatic necrosis virus, IPNV) derived from salmonids with IC50 values of about 10 micrograms/ml, 20 micrograms/ml and 32 micrograms/ml, respectively. The agent was not toxic for the host cells (chinook salmon embryo, CHSE-214) at the IC50 concentrations. Labeling of IHNV viral RNA and host cellular DNA and RNA with [3H]uridine and [3H]thymidine during drug treatment showed that guanine 7-N-oxide did not reduce the incorporation of these precusors into RNA and DNA. The anti-IHNV activity of guanine 7-N-oxide was enhanced synergistically by neplanocin A, an inhibitor of RNA methylation. The mechanism of action of guanine 7-N-oxide is discussed, in regard to maturation of viral messenger RNA including capping.     

扇贝裙边糖胺聚糖体外抗I型单纯疱疹病毒实验研究

目的研究扇贝裙边糖胺聚糖(glycosaminoglycan from Scallop Skirt,SS-GAG)体外抗单纯疱疹病毒Ⅰ型(HSV-Ⅰ)的作用。方法运用单纯疱疹病毒Ⅰ型(HSV-ⅠSM44株),并以非洲绿猴肾细胞(Vero细胞)为宿主细胞,通过观察病毒感染后的细胞变性反应(cytopathic effect,CPE)和运用MTT比色法,检测不同浓度药物SS-GAG对Ⅰ型单纯疱疹病毒是否有直接的灭活作用、对HSV-Ⅰ感染复制的抑制活性以及对HSV-Ⅰ感染细胞的综合作用等,并观察药物对Vero细胞的毒性作用。结果与病毒对照组相比,SS-GAG各浓度组(100、50、25mg.L-1)能有效地保护经HSV-Ⅰ感染的Vero细胞,使细胞活性增强(P<0.01);并减弱HSV-Ⅰ导致的病变效应,抑制病毒的复制。此作用随着药物浓度的增加而增强。但是SS-GAG对HSV-Ⅰ没有直接的灭活作用;SS-GAG在50~1600mg.L-1浓度范围内对Vero细胞无明显的细胞毒性。结论SS-GAG在体外实验系统中显示出明显的保护宿主细胞抵抗HSV-Ⅰ病毒感染的活性作用。     

Antiviral activity of uridine 5'-diphosphate glucose analogues against some enveloped viruses in cell culture

Twenty five analogues of uridine 5'-diphosphate glucose were screened against herpes simplex type 2, vaccinia virus, Sindbis virus and African swine fever virus. After screening, the compound 5'-[[[[(2",3",4",6"-tetra-O-benzoyl-alpha-D- glucopyranosyl)oxi]carbonyl]amino]sulfonyl]uridine (2), the synthesis of which has been reported (Camarasa et al., J. Med. Chem. 28, 40-46, 1985), was selected for further study. This compound showed in vitro activity against all viruses tested. The replication of herpes virus type 2 and African swine fever virus was completely inhibited at 100 micrograms/ml and 150 micrograms/ml respectively; vaccinia virus and Sindbis virus were inhibited to a lesser extent. The compound may inhibit several steps in the viral replication process.     

The HIV replication inhibitor 3'-fluoro-3'-deoxythymidine blocks sialylation of N-linked oligosaccharides.

The ability of 3'-fluoro-3'-deoxythymidine (FLT) to interfere with glycosylation was investigated in an experimental system, where the effects on the herpes simplex virus type 1-specified glycoprotein gC were determined. By adding FLT to HSV-infected cells after the peak of DNA synthesis, it was possible to segregate possible effects on nucleic acid metabolism from the effects on glycosylation of gC. It was found that FLT treatment of HSV-infected cells at concentrations of 20-500 micrograms/ml resulted in a significant increase in the electrophoretic mobility of gC, indicating a reduction of the amount of carbohydrates incorporated into gC. Lectin-binding assays demonstrated that the FLT treatment blocked addition of sialic acid to complex type N-linked glycans. The effects on glycosylation were observed in cells infected with an HSV mutant, deficient in thymidine kinase (TK), but not in cells infected with wild type virus. The cells infected with the wild type virus contained five times more total FLT metabolites than the cells infected with the TK-deficient mutant, whereas the latter cell type contained significantly higher amounts of unmetabolized FLT. This result indicates that FLT itself, and not a metabolite, was responsible for the effects on glycosylation.     

Indolocarbazoles exhibit strong antiviral activity against human cytomegalovirus and are potent inhibitors of the pUL97 protein kinase

We have analyzed a panel of protein kinase inhibitors (PKIs) and found that some indolocarbazoles (G?6976, K252a, K252c) proved to be highly effective inhibitors of GCV-sensitive and -resistant human cytomegalovirus (HCMV) strains, but did not show any effect against herpes simplex virus. Antiviral activity was determined by focus reduction assays (IC(50) ranging from 0.009 to 0.4 microM). Other inhibitors of serine/threonine kinases (G?6850, H-7, roscovitine) were found to be ineffective. Virus yield at 5 days after infection was reduced by three orders of magnitude with nanomolar concentrations of the indolocarbazoles. These compounds were fully effective when added up to 24 h post infection and showed reduced activity up to 72 h post infection. Cytotoxicity assays in proliferating and non-proliferating cells demonstrated that the effective antiviral concentration of these compounds was significantly lower than either antiproliferative (IC(50)/CC(50) ranging from 6.5 to 390) or cytotoxic (IC(50)/CC(50) ranging from 72. 5 to 1000) doses. The effects of PKIs on the virus-encoded protein kinase pUL97 were studied using recombinant vaccinia viruses. Indolocarbazoles strongly inhibited both pUL97 autophosphorylation (IC(50) ranging from 0.0012 to 0.013 microM) and pUL97-dependent ganciclovir phosphorylation (IC(50) ranging from 0.05 to 0.26 microM). Other inhibitors of serine/threonine kinases showed only weak (G?6850) or no (H-7, roscovitine) effect on these pUL97 functions, while oxoflavone tyrosine kinase inhibitors had no effect at all.     

7-Aminoquinolines. A novel class of agents active against herpesviruses

A series of 7-aminoquinoline derivatives was synthesized and evaluated for their capacity to produce cytotoxicity in KB cells and to inhibit the replication of herpes simplex virus (HSV) type 1. All compounds tested inhibited the replication of HSV-1 with 50% inhibitory concentrations in the range of 2-50 micrograms/mL. The antiviral activity of many compounds, however, was separated from cytotoxicity to replicating uninfected cells by only two- to fivefold higher than those required for antiviral activity. Nonetheless, six compounds (10, 28, 29, 32, 34, and 36) were identified in which the separation was greater than fivefold. All compounds examined were more potent inhibitors of viral DNA synthesis than the cellular DNA synthesis.     

Efficacy of (E)-5-(2-bromovinyl)-2'-deoxyuridine against different herpes simplex virus strains in cell culture and against experimental herpes encephalitis in mice

(E)-5-(2-Bromovinyl-2'-deoxyuridine (BrVUdR) showed strong antiviral activity against different laboratory strains and clinical isolates of herpes simplex virus type 1 (HSV-1) on primary rabbit testes (PRT) cells with a 50% inhibition of plaque formation (ID50) at 0.01-0.02 microM. One laboratory strain (HSV-1-S), however, was completely refractory even at concentrations as high as 100 microM. In contrast, the ID50S for all herpes simplex virus type 2 (HSV-2) strains were about 10(2) - 10(3) times higher (8-25 microM) than for the HSV-1 strains. No toxicity in mice treated with 140 mg BrVUdR/kg/day for 14 days was observed, and successful treatments of herpes encephalitis in mice induced experimentally by intracerebral infection with one laboratory strain (HSV-1-Kupka) and one clinical isolate (HSV-1-64) were achieved. Treatment of encephalitis in mice induced by the strain HSV-1-S insensitive to BrVUdR in cell culture failed to be effective. Similar antibody titers against HSV-1 were found in surviving mice of the control and of the BrVUdR-treated groups.